Yeast heterochromatin regulators Sir2 and Sir3 act directly at euchromatic DNA replication origins.

Most active DNA replication origins are found within euchromatin, while origins within heterochromatin are often inactive or inhibited. In yeast, origin activity within heterochromatin is negatively controlled by the histone H4K16 deacetylase, Sir2, and at some heterochromatic loci also by the nucleosome binding protein, Sir3. The prevailing view has been that direct functions of Sir2 and Sir3 are confined to heterochromatin. However, growth defects in yeast mutants compromised for loading the MCM helicase, such as cdc6-4, are suppressed by deletion of either SIR2 or SIR3. While these and other observations indicate that SIR2,3 can have a negative impact on at least some euchromatic origins, the genomic scale of this effect was unknown. It was also unknown whether this suppression resulted from direct functions of Sir2,3 within euchromatin, or was an indirect effect of their previously established roles within heterochromatin. Using MCM ChIP-Seq, we show that a SIR2 deletion rescued MCM complex loading at ~80% of euchromatic origins in cdc6-4 cells. Therefore, Sir2 exhibited a pervasive effect at the majority of euchromatic origins. Using MNase-H4K16ac ChIP-Seq, we show that origin-adjacent nucleosomes were depleted for H4K16 acetylation in a SIR2-dependent manner in wild type (i.e. CDC6) cells. In addition, we present evidence that both Sir2 and Sir3 bound to nucleosomes adjacent to euchromatic origins. The relative levels of each of these molecular hallmarks of yeast heterochromatin-SIR2-dependent H4K16 hypoacetylation, Sir2, and Sir3 -correlated with how strongly a SIR2 deletion suppressed the MCM loading defect in cdc6-4 cells. Finally, a screen for histone H3 and H4 mutants that could suppress the cdc6-4 growth defect identified amino acids that map to a surface of the nucleosome important for Sir3 binding. We conclude that heterochromatin proteins directly modify the local chromatin environment of euchromatic DNA replication origins.

The protein kinase C orthologue PkcA plays a role in cell wall integrity and polarized growth in Aspergillus nidulans.

The calC2 mutation in Aspergillus nidulans causes hypersensitivity to Calcofluor White, along with other drug sensitivities that indicate a defect in cell wall integrity. We have cloned CalC by complementation, isolating the A. nidulans orthologue of protein kinase C (PkcA). The pkcA allele of the calC2 strain contains a mutation predicted to introduce a charged arginine residue in place of neutral glycine at a conserved site located immediately beside the C1B regulatory domain. Both PkcA and calC2 map to the same region of chromosome VIII. A PkcA::GFP chimera localizes to hyphal apices and growing septa, as well as to the conidiogenous apices of phialides, indicating a role for PkcA in polarized cell wall growth. These observations support the hypothesis that the role of PkcA in A. nidulans, is comparable to that played by Pkc1p in the Saccharomyces cerevisiae cell wall integrity pathway.